Enterovirus B types cause severe infection in infants aged 0–3 months

Background Enterovirus (EV) infections are being increasingly seen in younger infants, often being more severe than in older children. The risk factors of EV infection in infants have been inadequately investigated till date. Methods We conducted a retrospective study on hospitalized children with laboratory-confirmed EV infection (50 infants aged 0–3 months and 65 older than 3 months) at a tertiary care center in China. Prevalence, clinical characteristics, and genetic features of the virus were analyzed, and independent predictors for severe infection were assessed. Results Clinical findings showed that severe infection was more common in infants aged 0–3 months than in older children (78.0% vs. 35.4%, p < 0.001), with higher morbidity of pneumonia, meningitis, and sepsis (p < 0.01). EV-B types were detected more frequently in infants aged 0–3 months than in older children (88.0% vs. 7.7%, p < 0.001). Echovirus 11 was the most identified EV-B, and it recombined with E6 in P2 and P3 regions. Risk factors for severe EV infection included EV-B types infection, age less than 3 months, elevated alanine aminotransferase level, abnormal platelet count, and abnormal cerebrospinal fluid characteristics. Conclusions Our data indicated that EV-B types mainly cause severe infection in infants aged 0–3 months. Therefore, knowledge about EV-B types could have implications in designing effective intervention and prevention strategies for young infants with severe EV infection. Supplementary Information The online version contains supplementary material available at 10.1186/s12985-023-01965-9.

E29-33, CVB1-6, and CVA9) are commonly identified in children less than 1 year old and are characterized by serious disease, with high mortality rate [10,11]. The circulation of EV-B types has been associated with several EV outbreaks in Asia, Europe, and the USA in recent years [12][13][14]. However, the incidence of infant infections and circulating types varies markedly across countries and regions.
There is growing interest in the understanding of epidemiology of EV in infants, particularly due to their association with severe disorders. Awareness of the clinical features associated with severe conditions in infants, recognition of the risk factors, and monitoring of the infection types might help pediatricians diagnose severe cases promptly and treat them appropriately to reduce mortality. In this study, we described the epidemiological, clinical, and genetic characteristics of EV infections in a cohort of children admitted to a tertiary care center in Guangzhou, China. Furthermore, with the clinical and molecular epidemiological patterns of EV infection, we aimed to explore the clinical spectrum and relationship of individual EV types with the risk factors of severe infection in infants.

Study design and patients
Children hospitalized with laboratory-confirmed EV infection, between January and December 2019, at the Guangdong Women and Children Hospital, Guangzhou, China were included in this study. A confirmed case of EV infection was defined by positive EV-RNA findings in stool, plasma, and/or cerebrospinal fluid (CSF) specimens. Moreover, we extracted demographic data, clinical symptoms or signs, laboratory data, CSF parameters, and outcomes upon admission from the patients' electronic medical records, and all laboratory tests were performed according to the clinical care needs of the children.

EV detection and type determination
Nucleic acids were extracted from stool, plasma, and/or CSF samples of suspected children using the Ex-DNA/ RNA Virus Kits (Tianlong Bio-technology, Suzhou, China), and amplified using the commercial pan-enterovirus q-PCR kit (DAAN Gene, Guangzhou, China) for EV in the 7500 fast real-time PCR system (Applied Biosystems, Foster City, USA), according to the manufacturer's instructions [15]. In addition, all EV-positive samples were amplified on the basis of partial VP1 sequences for further type identification, as described previously [15].

Full-length genome amplification
To understand the molecular epidemiology of EV in infants better, three long-distance PCR amplifications of the most common EV type in infants were obtained using the TaKaRa LA Taq ® Kit (Dalian, China). The primers used for RT-PCR are listed in Additional file 1: Table S1, and the full-length genome was sequenced using the "primer-walking" strategy. Finally, complete nucleotide sequences of selected strains were obtained by assembling the sequenced fragments, which were eventually submitted to GenBank (accession numbers: MW883610 to MW883614).

Phylogenetic and recombination analysis
Multiple sequence alignments were performed using the ClustalX, and phylogenetic trees were drawn using the neighbor-joining method via MEGA X [16]. Bootstrap analyses with 1000 re-samplings were performed to determine the confidence values for groupings within the phylogenetic trees. The SimPlot and Bootscan analyses were performed using SimPlot 3.5.1 with a 500-nucleotide window moving in steps of 20 nucleotides [17]. Evolutionary divergence of the 5ʹ UTR, four viral capsid proteins (VP1-VP4), and seven non-structural proteins (2A-2C and 3A-3D) between the selected strains and 64 reference strains from the GenBank was estimated via MEGA X using the Kimura 2-parameter model [16]. Heatmap of the 5ʹ UTR, VP1-VP4, 2A-2C, and 3A-3D genes was created based on evolutionary divergences.

Statistical analysis
Descriptive statistical analysis for population characteristics in the study and clinical findings was performed using SPSS 22.0. Categorical variables are reported as frequencies and percentages after comparison using the chi-square test or Fisher's exact test. The quantitative data are presented as the median (interquartile range, IQR) and were compared using the nonparametric rank-sum test (Kruskal-Wallis test). Odds ratio (OR) and 95% confidence intervals (95% CIs) were used to estimate the risk factors of severe infection using SPSS 22.0, a multiple logistic regression analysis.
Since the incidence and types of EV infection are influenced by seasonal cycles, we checked the monthly variation (Fig. 2a). A total of 109 (94.8%) hospitalizations occurred from March to October, and the number of cases peaked from April to June. However, there was a significant difference in seasonal changes in the occurrence of EV infection between infants aged 0-3 months and children older than 3 months. A peak (mainly caused by E11) was observed in the infant group from April to July, with no case thereafter. On the contrary, two peaks were observed in the number of cases in the children group. The first peak was unexpected, appearing in April; the second peak appeared in August.  [12][13][14][15][16][17][18][19][20][21][22][23][24][25][26][27], which was significantly less than the 16.5 months (IQR 13.0-26.0) observed for EV-A infections (p < 0.001).

Clinical analysis
Clinical case files of the 115 patients, with symptoms registered by the pediatrician upon hospital admission, were carefully analyzed, according to age and EV species (Table 1 and Additional file 2: Table S2 . However, presentation with fever, rash, tachycardia, coughing, vomiting, startle, and convulsions was significantly less common among infants aged 0-3 months than in children older than 3 months (all p < 0.01). Although the clinical features of infants aged 0-3 months were not typical, the median days of hospital stay were significantly longer than those of older children [13][14][15][16][17]  vs. 5 (7.7%), p < 0.001] than older children. Hepatitis occurred in six (5.2%) infants and two (1.7%) children, and all the cases were associated with EV-B infections.
A total of 62 (53.9%) cases were classified as having severe disease, of whom 39 (62.9%) were infants. Thus, the incidence of severe EV-B-associated infection was 73.5% (36 of 49), which was significantly higher than the 39.4% (26 of 66) of EV-A infections (p < 0.001). Notably, two EV-B-associated individuals died after the onset of the disease; one 5-day-old infant was infected with CVB3, and died of pulmonary edema and subventricular hemorrhage after 28 days of ventilator support, and another (7-month-old) was infected by E18, and died of brain edema.

Laboratory parameters of blood and CSF
Blood test results, according to age and EV serotypes, are presented in Table 2. Compared to older children, infants aged 0-3 months had higher neutrophil counts, lymphocyte counts, and lactate dehydrogenase (LDH) levels (all p < 0.05). Additionally, the levels of inflammatory markers like C-reaction protein (CRP) in older children were significantly higher than those in the infant group [15. CSF parameters according to EV type and age are shown in Table 2. In this study, a total of 60 CSF samples from 33 infants (aged 0-3 months) and 27 older children were included. CSF pleocytosis, elevated protein levels, and low CSF glucose were significantly more common in infants than in children (all p < 0.001). In addition, the positive rate of EV in CSF samples from young infants was 76.7% (23 of 30), which was significantly higher than the 20.0% (5 of 25) in the children group (p < 0.001).

Risk factors for severe infection
In this study, among the 115 patients with EV infections, 62 (53.9%) developed severe disease. Risk factors were analyzed between severe and non-severe EV infections by multiple logistic regression analysis using the forward stepwise logistic regression model. As shown in Table 3

Phylogenetic analysis of EV-B types
To understand the molecular epidemiology of EV-B types better, a portion of the VP1 gene from all 49 viruses isolated from EV-B infections was amplified and selected for phylogenetic analysis (Fig. 3). The phylogenetic tree indicated that all 30 E11 strains were closely related to viruses detected in China and the USA in 2018. Additionally, all six E18 strains showed high homology with the strains isolated from the Yunnan, Jiangsu, Hebei, and Sichuan provinces of China in 2015 and 2016.

Complete genome sequence analysis of E11
To help determine the evolution of predominant EV type in infants aged 0-3 months during the study year, we randomly performed full-length genome sequencing of E11 from five infants. Phylogenetic analysis revealed that all five E11 strains that circulated in Guangzhou city during 2019 clustered monophyletically with the E11 strains (MN597937 and MN597943) isolated from sewage samples in 2018 in Guangzhou city.
Based on the heatmap results of evolutionary divergence between E11 sequence and other closely related EV sequences, we found that the nucleotide sequences of 5ʹ UTR, VP1-VP4, 2A, and 2B regions of E11 strains displayed higher sequence identity with the strains (MN597927, MN597948, MN597926, and MN597950) isolated in Guangzhou city in 2018, whereas 2C and 3A-3D genes showed minimum evolutionary divergence with E6 strains from Jiangsu province (MK791151) and Zhejiang province (MN145871) of China in 2018  (Fig. 4a). The highest similarity of 2C and 3A-3D regions with E6 suggested possible recombination. Similarity plot and bootscanning were conducted between E11 and E6 strains to investigate the recombination phenomenon. Simplot analyses (Fig. 4b) showed that all five E11 strains in our study displayed the highest degree of identity with four E11 strains (MN597927, MN597948, MN597926, and MN597950) in the 5ʹ UTR and P1 (VP1-VP4) regions, and that similarity in the P2 (2B-2C) region decreased sharply. Thus, it was evident that recombination events with the E6 strain (MN597937 and MN597943) occurred partially in the P2 and P3 genomic regions. Furthermore, the Bootscan plot (Fig. 4c) indicated E11 strains in our study had a closer evolutionary relationship with E6 strains in the P2 and P3 genomic regions, with a sequence similarity of over 70.0%.

Discussion
This study showed that seasonal variations in EV infections with respect to age were evident. Most infants aged 0-3 months had a seasonal pattern of infections. They presented during summer, with a peak from April to July, which was apparently different from the epidemic features in older children, and consistent with previous studies [4,11,18]. Our current study also showed that the circulating EV types, affecting each age range, differed substantially. The most common EV detected in children older than 3 months was EV-A, whereas EV-B, represented by the E11 type, was significantly more frequent in younger infants. In the USA and Europe, EV-B is the one most commonly reported in neonates [12,13,19,20]. EV infection in neonates can present with clinical symptoms, ranging from asymptomatic, non-specific febrile illness to severe, life-threatening disease, and it is difficult to distinguish from non-EV infection based on the clinical signs only. In this study, fever was the most common symptom in both infants aged 0-3 months and older children. However, the other clinical symptoms, including rashes, tachycardia, coughing, vomiting, startle, and convulsions, frequently observed in children with HFMD, were not typical for infants, as suggested in earlier retrospective studies [13,21]. Rashes are often suggested as a diagnostic basis for viral infection. However, only 6.0-27.3% of cases of infants with EV infection have been reported with rashes [4,13,22]. In this study, only 8% (4/50) of the infants developed cutaneous rashes. As an important indication of host inflammation in viral infection, less frequent rashes make the early clinical diagnosis of EV infection difficult for neonatologists.
Severe illnesses due to EV are commonly seen in neonates; mortality is exceptionally high when meningitis, myocarditis, and hepatitis occur [4,13,23]. More than three-quarters of neonates were diagnosed with severe infection in this study, particularly in the first two weeks of life. Meningitis, sepsis, pneumonia, myocarditis, and/ or hepatitis are the most common clinical presentations. In contrast, only 35.4% of children older than 3 months showed severe infection. Meningitis is often associated with age-specific pleocytosis and/or neurological symptoms. Previous studies had shown that EVs can invade the central nervous system and disrupt the blood-brain barrier, resulting in more than 75% of meningitis cases, some of which are life-threatening [7,13,24]. In our study, out of 36 patients with meningitis, 28 (77.8%) were infants aged 0-3 months. It could be due to the immature brain of neonates and their imperfect blood-brain barrier. A higher detection rate of 76.7% (23 of 30) for EVs in CSF samples available from 30 neonates further substantiated this finding.
Pneumonia is the most common comorbidity in lower respiratory tract-EV-B infection, and can be rapidly progressive, leading to severe pneumonia [25]. In this study, more than one-third (44/115) cases were accompanied by pneumonia. EV-B types were the predominant pathogens in patients with pneumonia. Sepsis is a lifethreatening organ dysfunction caused by a dysregulated host response to infection associated with a wide range of pathogens, and pediatricians frequently encounter it in young infants [13,19,26]. Hepatitis is often associated with EV-B infections, and mortality is especially high when it occurs concomitantly with myocarditis [27]. In this study, a total of 8 cases (6 infants and 2 children) developed symptoms of hepatitis during hospitalization, and all were associated with EV-B infection.
Understanding the risk factors and monitoring the parameters associated with severe infection may lead to effective prophylaxis and prompt aggressive treatment to reduce morbidity and mortality. Infection with EV-B and younger age are considered the major risk factors for developing severe infections [5]. A retrospective study on 2356 infants with known EV types during 1983-2003 reported that CVB1-5 and E11 have an increased risk of neonatal infection, with CVB4 being associated with the highest mortality rate of 40% [28]. In our study, we found that EV-B infection and age less than 3 months increased the risk of EV infection 4.260-fold and 6.474-fold, respectively. Moreover, of the two deceased children, one was positive for CVB3 and the other was for E18, which illustrated that EV-B infection is a major risk factor. Notably, the mother of the 7-month-old child was also positive for E18 as per stool sample test, with 100% similarity in VP1 gene compared to that of the children, but showed no obvious clinical symptom. Previous studies had shown that transmission of EVs from mother to infant is relatively common, occurring in 30-50% of cases [23,29,30].
Xiao-Qing Lv et al. had previously reported that an abnormal platelet count could be an independent predictor of severe EV infection [31]. Our results also showed that if the platelet count was abnormal, the risk of severe infection was increased 2.745-fold. Furthermore, abnormal CSF test result, such as pleocytosis, constituted a significant risk factor for developing severe EV infection, as observed by previous investigators [32], and elevated protein levels and pleocytosis in CSF increased the risk of severe infection 13.913-fold and 9.481-fold, respectively. Moreover, we elucidated that the positive result of EV in CSF was an independent predictor of severe infection, and the OR increased to 12.071.
Genomic recombination is a major driving force in the evolution, diversification, and shaping of genetic architecture of EVs [33], and time-correlated recombination events of EV-B are more frequent than those of other human EV species [34]. However, until recently, only limited complete genome sequences of E11 strains were available in the public database, with most of them coming from non-clinical isolates. In this study, we obtained 5 full-length E11 genomes, analyzed their phylogenetic characteristics, and found homologous recombination events to have occurred with E6 strains in China in 2018. Multiple phylogenetic studies presented previously provided evidence that RNA recombination of EVs only occurred throughout the entire non-structural region, and recombination sites were mainly located in region P2 [33][34][35][36]; notably, the recombination site was in the junction between 2B and 2C, as per our study. These observations suggested that non-structural proteins may be functionally interchangeable with other variants within EVs. Furthermore, in the infected host, effective recombination events are critical for RNA viruses to overcome tissue-type specific antiviral selection, establish robust infection and virulence, and adapt rapidly to dynamic selective environments [37,38]. However, the changes in phenotypic characteristics of E11 recombination, including their fitness and pathogenicity, need to be investigated further.

Conclusions
In summary, EV affects infants aged 0-3 months differently and more severely than in older children. Clinical manifestations in infants with EV mainly included meningitis, sepsis, pneumonia, and even death. EV-B types were the most common in neonatal EV infection, and recombination events were observed in the P2 and P3 regions of predominant type E11 with E6 from China. In addition, we identified independent predictors of severe EV infection, including EV-B infection, age less than 3 months, elevated ALT level, abnormal platelet count, and abnormal CSF characteristics. Taken together, EV-B infections should be routinely considered in neonates with meningitis, sepsis, and pneumonia, with or without a rash, particularly during EV season.